The invention is in the field of sample analysis to determine the presence therein of Human Papilloma Virus (HPV) genotypes by amplifying HPV DNA present in the sample with a nucleic acid amplification process, e.g. the Polymerase Chain Reaction (PCR), using general primers (GPs). More in particular, the invention relates to an analysis of cervical smears which allows cervical carcinoma-related diagnosis and prognosis wherein the analysis comprises a GP-nucleic acid amplification process, e.g. GP-PCR, to determine whether the sample contains any HPV, followed by a typing of the HPV genotype present.
HPV comprises over 70 different epitheliotropic genotypes of which over 30 are mucosotropic. Approximately one third of these mucosotropic HPV genotypes have been isolated from or associated with cervical carcinomas (De Villiers, 1989; zur Hausen, 1991).
The PCR method has been introduced as the most sensitive method for the detection of HPV DNA in clinical specimens. However, a significant heterogeneity at the nucleotide level is found between the different HPV genotypes. This has hampered the development of a simple universal PCR test for the detection of all HPV genotypes. Despite this, HPV-PCR methods have been developed which allow the detection of a broad spectrum of mainly mucosotropic HPV genotypes (Manos et al., 1989; Gregoire et al., 1989; Snijders et al., 1990).
A combination of the general primers GP5 and GP6, originally selected from the HPV L1 region on the basis of sequence information of HPV6, HPV11, HPV16, HPV18, HPV31 and HPV33 (Snijders et al., 1990; WO 91/10675), was found to amplify target DNA of at least 27 mucosotropic HPV genotypes under conditions that allow mismatch acceptance (Van den Brule et al., 1990a, 1992; de Roda Husman et al., 1994a). The strength of this GP5/6-mediated PCR method has been substantiated further by the detection of HPV DNA in 100% of cervical scrapes classified cytomorphologically as Pap IV (carcinoma in situ) and Pap V (carcinoma) in the Netherlands (Van den Brule et al., 1991; de Roda Husman et al., 1994a). This suggests that in the Dutch population all genital high risk HPVs can be detected by this assay.
Still, using GP-PCR in routine diagnostic practice, it has been found that a small number of clinical samples gives rise to ambiguous results, reflected by GP-PCR signals that are weaker than signals obtained from 50-100 Siha cells (which contain one copy of HPV16 per cell; Van den Brule et al., 1990a). This may complicate interpretation of screening results since it is presently unknown whether the weak signals represent a cross-reaction with cellular sequences or the presence of HPV genotypes which show a reduced sensitivity in the GP-PCR. It has been shown previously that some HPV types like HPV30 are detected with a decreased sensitivity in the GP-PCR (Snijders et al., 1990), and also the recently sequenced HPV types HPV39 and HPV51, showing more than three mismatches with one of the primers, have revealed a reduction in GP-PCR sensitivity (data not shown). Furthermore, some HPV types (e.g. HPV18) give rise to additional bands in the GP5/6 PCR (Snijders et al., 1990).
Recently, several groups have found that despite the presence of primer/template mismatches, a successful amplification by PCR can be ensured by the presence of perfectly matching nucleotides at the 3xe2x80x2-ends of the primers (Newton et al., 1989; Sommer and Tautz, 1989; Evander and Wadell, 1991).
Moreover, it also has been found that increased primer length contributes to a more efficient amplification, probably by increasing the stability of the primer/template complex (Mack and Sninsky, 1988).
Sequence analysis of the GP5/6 PCR products of different HPV genotypes has revealed the presence of HPV-specific amino acid consensus sequences directly adjacent to the 3xe2x80x2-ends of GP5 and GP6 (Van den Brule et al., 1992). We investigated the utility of GP5/6 primers elongated with highly conserved sequences at their 3xe2x80x2-ends. These elongated primers (named GP5+ and GP6+) were tested in the PCR using a model system of cloned HPV DNAs and subsequently evaluated on cervical smears which previously showed ambiguous or negative results with the original GP5/6 assay.
The results surprisingly revealed that an elongation of GP5 and GP6 with conserved sequences at their 3xe2x80x2-ends can overcome reduced PCR efficiencies most likely related to the number of primer/target mismatches and increase primer-template stability. Moreover, the use of elongated GP5/6 in the PCR resulted in the clarification of HPV status in cytomorphologically normal cervical scrapes which previously showed ambiguous or negative GP-PCR results.
Another desideratum in the field of HPV detection is a means to differentiate quickly between high risk and low risk HPV types. So far, individual HPV typing has been performed on the products of nucleic acid amplification by hybridization analysis using HPV type-specific oligonucleotide probes or probes consisting of cloned HPV types, or by additional type-specific PCRs. This kind of analysis entails much work, especially if one considers that the clinician usually wants to know only whether there is a high or low risk of cervical cancer. It is known by now that only a restricted group of 15 HPV types (Nos. 16, 18, 31, 33, 35, 39, 45, 51, 52, 54, 56, 58, 59, 66 and 68) is associated with cervical carcinomas and carcinomas in situ (see the review of De Villiers, 1989). In a recent study it was found that 10 different HPV types (Nos. 6, 16, 18, 31, 33, 45, 51, 52, 54 and 58) were present in PAP IV scrapes tested by GP-PCR (De Roda Husman et al., 1994b). Furthermore, preliminary results from follow up studies show that only high risk HPV types show progression from cytologically normal cervix to cervical intraepithelial neoplasia (CIN) III. Classification into HPV groups with different biological behaviour instead of individual HPV typing would be less confusing and will be appreciated by the clinician. HPV detection assays using a panel of high risk HPV probes will detect most HPV-induced carcinomas and carcinomas in situ. So, for the sake of an early detection of cervical cancer, there is a need for HPV detection assays permitting a rapid differentiation between all known high risk and low risk HPV types.
We herein describe the design and performance of type-specific oligonucleotide probes which may be used either separately or in the form of cocktails for screening the GP5+/6+ mediated DNA amplification products on high and low risk HPV genotypes.
Sequence analysis of HPV GP5/6-mediated PCR products has revealed the presence of short highly conserved sequences adjacent to the 3xe2x80x2-ends of both primers. Since perfect matching of 3xe2x80x2 primer ends is critical for an efficient PCR and elongation of primers gives an additional stabilization of primer/template complexes, part of these sequences were used to elongate GP5 and GP6 at their 3xe2x80x2-ends. Using reconstruction experiments with different molecularly cloned HPVs, the elongated primers (named GP5+ and GP6+) showed a clearly improved detection of especially HPV genotypes having more than 3 mismatches with one or both primers. The strength of the method was further substantiated by improved HPV detection in cytomorphologically normal cervical scrapes which showed ambiguous results in the original HPV GP5/6-mediated PCR. Also a small percentage of cytological normal scrapes which were originally HPV-negative with HPV GP5/6-mediated PCR became positive after application of the elongated GP5/6 primers.
Therefore, the invention provides a general primer pair GP5/6 which has been elongated at the 3xe2x80x2-ends with adjacent highly conserved sequences thereby improving HPV detection in cervical smears.
Furthermore, by computer-assisted sequence analyses of the amplication product obtained by GP5/6 and GP5+/6+ PCR, which amplification product has a length of about 150 bp, we selected (from the internal part of the GP5/6 region) 30-mer oligonucleotides specific for 24 different HPV genotypes. These new oligonucleotides, suitably labeled with e.g. digoxygenine, proved useful as HPV-specific probes in Southern blot analysis of high copy PCR products derived from the same HPV types. No cross-hybridisations were found. We made two cocktails which enabled a specific and sensitive differentiation between HPV types of high risk (Nos. 16, 18, 31, 33, 35, 39, 45, 51, 52, 54, 56 and 58) and low risk (Nos. 6, 11, 34, 40, 42, 43 and 44) for the development of cervical cancer. These probe cocktails may be succesfully applied for a rapid identification of high risk HPV types in GP-PCR based HPV screening of cervical scrapes.
The invention provides an oligonucleotide selected from the group consisting of
(i) the 23-mer 5xe2x80x2-TTTGTTACTGTGGTAGATACTAC-3xe2x80x2 (SEQ ID NO:1) or the 23-mer which is complementary to SEQ ID NO:1;
(ii) a 23-mer derived from (i) by from 1 to 5 nucleotide substitutions;
(iii) a 23+-mer having a 3xe2x80x2 terminal sequence consisting of (i) or (ii);
(iv) a fragment of (i) or (ii) having a length of from 8 to 18 nucleotides.
The oligonucleotide (i) is derived from a relatively conserved part of the L1 region of HPV. The 23-mer of SEQ ID NO:1 is composed of the primer GP5, as described in WO91/10675, and an extension at the 3xe2x80x2 terminus of the three additional nucleotides TAC. In view of said 3xe2x80x2 extension, it is referred to herein as GP5+.
The invention also includes the complementary sequence, which may be useful in certain kinds of nucleic acid amplification methods, such as in the LCR (Ligase Chain Reaction; see Barany 1991). In view of the possibility to use LCR technology, the invention also includes fragments of (i) which contain from 8 to 18 nucleotides. Preferably, said fragments correspond to either the 5xe2x80x2-end or the 3xe2x80x2-end of (i). The invention also includes such fragments of from 8 to 18 nucleotides of (ii), (v), (vi), (viii), (ix) and (x), which will be described furtheron.
In the LCR, a thermostable ligase is used for a cyclic joining of two oligonucleotides that are substantially adjacent to each other. xe2x80x9cSubstantially adjacentxe2x80x9d means that the distance between the two oligonucleotides is sufficiently small to allow the ligase enzyme to ligate the two oligonucleotides. Preferably, the two oligonucleotides are immediately adjacent to each other. LCR cycli consist of denaturation, annealing and ligation steps similar to the PCR. Thus, a newly formed oligonucleotide after ligation serves as a target for the annealing and ligation of the complementary oligonucleotides and, consequently, an exponential enrichment can be achieved.
With respect to HPV detection, LCR technology may be used. By using as primers in said LCR, one of the general primers of this invention, together with an oligonucleotide complementary thereto, plus a primer based on an adjacent sequence, together with an oligonucleotide complementary thereto, it is possible, depending on the choice of these latter primer sequences, to make amplification dependent on the presence of particular HPV types or groups of HPV types.
Alternatively, LCR may be carried out using a pair of general primers of this invention plus oligonucleotides complementary thereto, wherein the pair of general primers of this invention consists of two different fragments of the same general primer. Preferably, one fragment corresponds to the 5xe2x80x2-end of said general primer and the other fragment corresponds to the 3xe2x80x2-end of said general primer, and the two fragments do not overlap but are substantially adjacent sequences. The fragments should have a length of from 8 to 18 nucleotides. An LCR based on the use of such fragments of the same general primer is useful for the detection of genital HPV genotypes in general.
The oligonucleotide (ii) is derived from SEQ ID NO:1, or from its complementary sequence, by from 1 to 5 nucleotide substitutions. Preferably, said substitutions concern substitutions occurring between different HPV strains. For example, the 4th nucleotide (G) is substituted in some strains by C (in HPV32, HPV39 and HPV57), in others by T (in HPV42), in others by A (in HPV51). Therefore, the invention covers oligonucleotides which contain any one of these, or similar substitutions.
Preferably, however, nucleotide substitutions which give rise to self-annealing or hair-pin loop formation of the oligonucleotide molecules are avoided. For example, substitution of the 21st nucleotide (T) by C is less preferred as the resulting molecule is prone to self-annealing or hair-pin loop formation, for example as follows:
The oligonucleotide (iii) is a 23+-mer, i.e. an oligonucleotide of more than 23 nucleotides. The 3xe2x80x2 terminal sequence consists of oligonucleotide (i) or oligonucleotide (ii). The extension at the 5xe2x80x2-end may have any length. Preferably, however, the total length of the oligonucleotide is not more than 50 nucleotides, more preferably not more than 40 nucleotides. Shorter oligonucleotides, such as those consisting of exactly 23 nucleotides, can be prepared more readily in good yield, whereas longer oligonucleotides may be preferable in view of higher efficiency in the PCR, or because the added sequence at the 5xe2x80x2-end brings a practical advantage.
For example, the added sequence at the 5xe2x80x2-end may comprise one or more restriction enzyme recognition sequences (restriction sites) such as, for example, BamHI, EcoRI and HindIII sites. Such primers are identified herein as Res primers. In addition to the advantage of a higher efficiency in the PCR, these extended oligonucleotides have the practical advantage of facilitating direct cloning of the amplimers obtained in the PCR, for instance into the plasmid pBR322 and into plasmids derived therefrom, such as p.Gemini vectors. Thus, the amplimers can be made suitable for conventional double-stranded sequencing (cloning capacity of 100 bp to several kb). The amplimers can also be cloned into the phage M13 (mp 18 and 19) for single-stranded sequencing (cloning capacity 100-500 bp). Owing to rapid developments in the field of sequencing techniques, direct sequencing of the amplimers is also one of the options. Direct sequencing of the amplimer products even seems to be the best identification for the virus. See WO91/10675 which, in its entirety, is incorporated herein by reference.
As another example, the added sequence at the 5xe2x80x2-end may comprise a promoter sequence such as, for example, a T7 promoter sequence which is:
5xe2x80x2-AAT TCT AAT ACG ACT CAC TAT AGG GGG A -3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:26);
or a T3 promoter sequence which is:
5xe2x80x2-TTA TTA ACC CTC ACT AAA GGG AAG -3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:27);
or a SP6 promoter sequence which is:
5xe2x80x2-ATT TAG GTG ACA CTA TAG AAT AC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:28).
Such primers, which may include an insertion of one or more nucleotides between the promoter sequence and the 3xe2x80x2 terminal sequence (consisting of oligonucleotide (i) or oligonucleotide (ii)), e.g. to improve the activity of the polymerase used, are identified herein as Pol primers. These primers have the added advantage of allowing the start of RNA polymerases to synthesize RNA species. Thereby, such primers allow the amplification of target nucleic acid sequences as RNA molecules. Said RNA molecules can be used in RNA amplification systems, such as NASBA(trademark) (nucleic acid sequence based amplification).
The NASBA method (see Kievits et al., 1991) is an isothermal method for the amplification of target RNA or DNA accomplished by the simultaneous enzymatic activity of a reverse transcriptase such as AMV reverse transcriptase, an RNA polymerase such as T7 RNA polymerase, and an RNase such as RNase H. For example, an RNA NASBA comprises an extension of a forward (or backward) primer which contains a T7, T3 or SP6 promoter sequence by reverse transcriptase (RT) on an RNA/DNA template, degradation of the RNA strand by RNase H (or heat denaturation for dsDNA which is formed in the case of a DNA NASBA), synthesis of a second DNA strand by a backward (or forward) primer extension with AMV-RT and RNA synthesis by T7, T3 or SP6 RNA polymerase. With RNA synthesis, the system enters the cyclic phase which is based on the above principles.
For HPV detection purposes, the forward primers can be either Pol GP5+ or Pol GP6+ primers while GP6+ and GP5+, respectively, can be used as the backward primers.
The invention also provides an oligonucleotide selected from the group consisting of
(v) the 25-mer 5xe2x80x2-GAAAAATAAACTGTAAATCATATTC-3xe2x80x2 (SEQ ID NO:2) or the 25-mer which is complementary to SEQ ID NO:2;
(vi) a 25-mer derived from (iv) by from 1 to 5 nucleotide substitutions;
(vii) a 25+-mer having a 3xe2x80x2 terminal sequence consisting of (iv) or (v);
(viii) the 28-mer 5xe2x80x2-GAAAAATAAACTGTAAATCATATTCTTC-3xe2x80x2 (SEQ ID NO:10) or the 28-mer which is complementary to SEQ ID NO:10;
(ix) the 28-mer 5xe2x80x2-GAAAAATAAACTGTAAATCATATTCCTC-3xe2x80x2 (SEQ ID NO:18) or the 28-mer which is complementary to SEQ ID NO:18;
(x) a 28-mer derived from (vii) or (viii) by from 1 to 5 nucleotide substitutions;
(xi) a 28+-mer having a 3xe2x80x2 terminal sequence consisting of (vii), (viii) or (ix);
(xii) a fragment of (v), (vi), (viii), (ix) or (x) having a length of from 8 to 18 nucleotides.
The oligonucleotide (v) is derived from a relatively conserved part of the L1 region of HPV. The 25-mer of SEQ ID NO:2 is composed of primer GP6, as described in WO91/10675, and an extension at the 3xe2x80x2 terminus of the five additional nucleotides TATTC. In view of this 3xe2x80x2 extension, it is referred to herein as GP6+.
The invention includes the complementary sequence, which may be useful in nucleic acid amplification methods such as the LCR.
The same applies to fragments having a length of from 8 to 18 nucleotides.
The oligonucleotide (vi) is derived from SEQ ID NO:2 or from its complementary sequence by from 1 to 5 nucleotide substitutions. Preferably, as in the case of oligonucleotide (ii), said substitutions concern substitutions occurring between different HPV strains. For example, the 11th nucleotide (C) is substituted in many strains by T (in HPV6B, HPV13, HPV31, HPV39, HPV42, HPV51, HPV52, HPV53 and HPV56). The 21st nucleotide (T) is substituted by A in several strains (in HPV11, HPV13, HPV31 and HPV52). The 23rd nucleotide (T) is substituted by C in several strains (in HPV6B, HPV11, HPV39 and HPV51). Therefore, the invention covers oligonucleotides containing these, and similar substitutions, such as, for example, the 25-mers:
5xe2x80x2-GAAAAATAAACTGTAAATCAAATTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:3)
5xe2x80x2-GAAAAATAAACTGTAAATCATACTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:4)
5xe2x80x2-GAAAAATAAACTGTAAATCAAACTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:5)
5xe2x80x2-GAAAAATAAATTGTAAATCATATTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:6)
5xe2x80x2-GAAAAATAAATTGTAAATCAAATTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:7)
5xe2x80x2-GAAAAATAAATTGTAAATCATACTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:8)
5xe2x80x2-GAAAAATAAATTGTAAATCAAACTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:9)
Preferably, however, nucleotide substitutions which give rise to self-annealing of the oligonucleotide molecules are avoided, as explained above for oligonucleotide (ii).
Oligonucleotide (viii) is the 28-mer of SEQ ID NO:10 which is composed of SEQ ID NO:2 and a 3xe2x80x2 extension consisting of the three nucleotides TTC, or the complementary sequence of SEQ ID NO:10, and oligonucleotide (ix) is the 28-mer of SEQ ID NO:18 which is composed of SEQ ID NO:2 and a 3xe2x80x2 extension consisting of the three nucleotides CTC, or the complementary sequence of SEQ ID NO:18. The additional extension at the 3xe2x80x2-end is possible because the relevant conserved part of the L1 region encompasses a further glutamic acid codon.
The oligonucleotide (x) is derived from SEQ ID NO:10, or from its complementary sequence, or from SEQ ID NO:18, or from its complementary sequence, by from 1 to 5 nucleotide substitutions. Preferably, as in the case of oligonucleotides (ii) and (vi), said substitutions concern substitutions occurring between different HPV strains. Examples of oligonucleotide (x) are:
5xe2x80x2-GAAAAATAAACTGTAAATCATATTCTTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:10)
5xe2x80x2-GAAAAATAAACTGTAAATCAAATTCTTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:11)
5xe2x80x2-GAAAAATAAACTGTAAATCATACTCTTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:12)
5xe2x80x2-GAAAAATAAACTGTAAATCAAACTCTTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:13)
5xe2x80x2-GAAAAATAAATTGTAAATCATATTCTTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:14)
5xe2x80x2-GAAAAATAAATTGTAAATCAAATTCTTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:15)
5xe2x80x2-GAAAAATAAATTGTAAATCATACTCTTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:16)
5xe2x80x2-GAAAAATAAATTGTAAATCAAACTCTTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:17)
5xe2x80x2-GAAAAATAAACTGTAAATCATATTCCTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:18)
5xe2x80x2-GAAAAATAAACTGTAAATCAAATTCCTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:19)
5xe2x80x2-GAAAAATAAACTGTAAATCATACTCCTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:20)
5xe2x80x2-GAAAAATAAACTGTAAATCAAACTCCTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:21)
5xe2x80x2-GAAAAATAAATTGTAAATCATATTCCTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:22)
5xe2x80x2-GAAAAATAAATTGTAAATCAAATTCCTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:23)
5xe2x80x2-GAAAAATAAATTGTAAATCATACTCCTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:24)
5xe2x80x2-GAAAAATAAATTGTAAATCAAACTCCTC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:25)
Oligonucleotide (vii) is a 25+-mer having a 3xe2x80x2 terminal sequence consisting of (v) or (vi); and oligonucleotide (xi) is a 28+-mer having a 3xe2x80x2 terminal sequence consisting of (viii), (ix) or (x). As in the case of oligonucleotide (iii), the extension at the 5xe2x80x2-end may have any length, but the total length of the oligonucleotide is preferably kept at 50 nucleotides at most, more preferably at not more than 40 nucleotides. It is preferred that the extension at the 5xe2x80x2-end comprises one or more restriction sites (Res primers) or a promoter sequence (Pol primers).
The invention further provides a pair of primers for use in a nucleic acid amplification process, such as PCR or NASBA, for the amplification of DNA of genital HPV genotypes, wherein the first primer consists of an oligonucleotide selected from the group consisting of (i), (ii) and (iii), and the second primer consists of an oligonucleotide selected from the group consisting of (v), (vi), (vii), (viii), (ix), (x) and (xi).
The invention also provides a primer set for use in a nucleic acid amplification process, such as LCR, for the amplification of DNA of genital HPV genotypes, wherein a first primer consists of an oligonucleotide selected from the group consisting of (i), (ii), (iii), (iv), (v), (vi), (vii), (viii), (ix), (x), (xi) and (xii), a second primer consists of an oligonucleotide complementary to said first primer, a third primer consists of an oligonucleotide corresponding to a region in the HPV genome substantially adjacent to the region from which said first primer is derived, and a fourth primer consists of an oligonucleotide which is complementary to said third primer.
The invention is further embodied in a method of amplifying DNA of genital HPV genotypes by means of a nucleic acid amplification process, comprising using a primer consisting of an oligonucleotide selected from the group consisting of (i), (ii), (iii), (iv), (v), (vi), (vii), (viii), (ix), (x), (xi) and (xii).
More particularly, the invention provides a method of amplifying DNA of genital HPV genotypes by means of a PCR using a pair of primers wherein a first primer consists of an oligonucleotide selected from the group consisting of (i), (ii) and (iii), and the second primer consists of an oligonucleotide selected from the group consisting of (v), (vi), (vii), (viii), (ix), (x) and (xi).
Similarly, the invention provides a method of amplifying DNA of genital HPV genotypes by means of a NASBA, using a pair of primers wherein a first primer consists of an oligonucleotide selected from the group consisting of (i), (ii) and (iii), and the second primer consists of an oligonucleotide selected from the group consisting of (v), (vi), (vii), (viii), (ix), (x) and (xi), with the proviso that one of the primers has a 5xe2x80x2-end which comprises a promoter sequence.
Also, the invention provides a method of amplifying DNA of genital HPV genotypes by means of an LCR, using a set of primers which comprises a first primer consisting of an oligonucleotide selected from the group consisting of (i), (ii), (iii), (iv), (v), (vi), (vii), (viii), (ix), (x), (xi) and (xii), a second primer consisting of an oligonucleotide which is complementary to the first primer, a third primer which consists of an oligonucleotide corresponding to a region in the HPV genome substantially adjacent to the region from which the first primer is derived, and a fourth primer consisting of an oligonucleotide which is complementary to the third primer.
The invention is also embodied in a method of analysing a sample, such as a cervical smear, for the presence therein of genital HPV genotypes which comprises amplifying DNA of a genital HPV present in the sample by means of a nucleic acid amplification process, employing a primer consisting of an oligonucleotide selected from the group consisting of (i), (ii), (iii), (iv), (v), (vi), (vii), (viii), (ix), (x), (xi) and (xii), and subsequently detecting a product of the amplification. Again, the nucleic acid amplification process may consist of, e.g., PCR, NASBA or LCR. The primers to be used in the amplification process should be appropriately chosen, depending on the kind of amplification process.
According to the invention, the primer annealing step in the nucleic acid amplification process is carried out preferably at a temperature of 30-50xc2x0 C., more preferably at a temperature of 35-45xc2x0 C., most preferably at a temperature of 38-42xc2x0 C.
Further, according to the invention, the nucleic acid amplification process is preferably carried out at a Mg2+ concentration of 2-10 mM, more preferably at a Mg2+ concentration of 2.5-5 mM, most preferably at a Mg2+ concentration of 3.0-4.0 mM.
According to the invention, the optimum results are obtained at an annealing temperature of about 40xc2x0 C. (normally 55xc2x0 C.) and at a Mg2+ concentration of about 3.5 mM (normally 1.5 mM Mg2+).
The new primers according to the invention enable the detection not only of genital HPV types whose sequence is already known, but also of HPV types whose sequence is (as yet) unknown and even of new HPV types.
According to the invention, it is preferred to adopt the general strategy for screening cervical smears which has been disclosed in WO91/10675. Said strategy is based on the combined use of HPV general primers according to the invention and the previously described HPV type-specific anticontamination primers. The procedure of this PCR strategy is described in WO91/10675 which has been incorporated herein by reference.
According to another aspect of this invention, the product of the amplification is detected by means of a DNA hybridization process using HPV type-specific oligonucleotide probes, the oligonucleotides of the probes being selected from the group consisting of:
5xe2x80x2-ATCCGTAACTACATCTTCCACATACACCAA-3xe2x80x2, (SEQ ID NO:29), specific for HPV-6;xe2x80x83xe2x80x83(a)
5xe2x80x2-ATCTGTGTCTAAATCTGCTACATACACTAA-3xe2x80x2, (SEQ ID NO:30), specific for HPV-11;xe2x80x83xe2x80x83(b)
5xe2x80x2-GTCATTATGTGCTGCCATATCTACTTCAGA-3xe2x80x2, (SEQ ID NO:31), specific for HPV-16;xe2x80x83xe2x80x83(c)
5xe2x80x2-TGCTTCTACACAGTCTCCTGTACCTGGGCA-3xe2x80x2, (SEQ ID NO:32), specific for HPV-18;xe2x80x83xe2x80x83(d)
5xe2x80x2-AGTACATTATCTGCAGCATCTGCATCCACT-3xe2x80x2, (SEQ ID NO:33), specific for HPV-26;xe2x80x83xe2x80x83(e)
5xe2x80x2-TGTTTGTGCTGCAATTGCAAACAGTGATAC-3xe2x80x2, (SEQ ID NO:34), specific for HPV-31;xe2x80x83xe2x80x83(f)
5xe2x80x2-TTTATGCACACAAGTAACTAGTGACAGTAC-3xe2x80x2, (SEQ ID NO:35), specific for HPV-33;xe2x80x83xe2x80x83(g)
5xe2x80x2-TACACAATCCACAAGTACAAATGCACCATA-3xe2x80x2, (SEQ ID NO:36), specofic for HPV-34;xe2x80x83xe2x80x83(h)
5xe2x80x2-GTCTGTGTGTTCTGCTGTGTCTTCTAGTGA-3xe2x80x2, (SEQ ID NO:37), specific for HPV-35;xe2x80x83xe2x80x83(i)
5xe2x80x2-TCTACCTCTATAGAGTCTTCCATACCTTCT-3xe2x80x2, (SEQ ID NO:38), specific for HPV-39;xe2x80x83xe2x80x83(j)
5xe2x80x2-GCTGCCACACAGTCCCCCACACCAACCCCA-3xe2x80x2, (SEQ ID NO:39), specific for HPV-40;xe2x80x83xe2x80x83(k)
xe2x80x835xe2x80x2-CTGCAACATCTGGTGATACATATACAGCTG-3xe2x80x2, (SEQ ID NO:40), specific for HPV-42;xe2x80x83xe2x80x83(l)
5xe2x80x2-TCTACTGACCCTACTGTGCCCAGTACATAT-3xe2x80x2, (SEQ ID NO:41), specific for HPV-43;xe2x80x83xe2x80x83(m)
5xe2x80x2-GCCACTACACAGTCCCCTCCGTCTACATAT-3xe2x80x2, (SEQ ID NO:42), specific for HPV-44;xe2x80x83xe2x80x83(n)
5xe2x80x2-ACACAAAATCCTGTGCCAAGTACATATGAC-3xe2x80x2, (SEQ ID NO:43), specific for HPV-45;xe2x80x83xe2x80x83(o)
5xe2x80x2-AGCACTGCCACTGCTGCGGTTTCCCCAACA-3xe2x80x2, (SEQ ID NO:44), specific for HPV-51;xe2x80x83xe2x80x83(p)
5xe2x80x2-TGCTGAGGTTAAAAAGGAAAGCACATATAA-3xe2x80x2, (SEQ ID NO:45), specific for HPV-52;xe2x80x83xe2x80x83(q)
5xe2x80x2-TACAGCATCCACGCAGGATAGCTTTAATAA-3xe2x80x2, (SEQ ID NO:46), specific for HPV-54;xe2x80x83xe2x80x83(r)
5xe2x80x2-GTACTGCTACAGAACAGTTAAGTAAATATG-3xe2x80x2, (SEQ ID NO:47), specific for HPV-56;xe2x80x83xe2x80x83(s)
5xe2x80x2-ATTATGCACTGAAGTAACTAAGGAAGGTAC-3xe2x80x2, (SEQ ID NO:48), specific for HPV-58;xe2x80x83xe2x80x83(t)
5xe2x80x2-TCTACTACTGCTTCTATTCCTAATGTATAC-3xe2x80x2, (SEQ ID NO:49), specific for HPV-59;xe2x80x83xe2x80x83(u)
5xe2x80x2-TACTGCTACATCCCCCCCTGTATCTGAATA-3xe2x80x2, (SEQ ID NO:50), specific for HPV-61;xe2x80x83xe2x80x83(v)
5xe2x80x2-TATTAATGCAGCTAAAAGCACATTAACTAA-3xe2x80x2, (SEQ ID NO:51), specific for HPV-66;xe2x80x83xe2x80x83(w)
5xe2x80x2-TCTACTACTACTGAATCAGCTGTACCAAAT-3xe2x80x2, (SEQ ID NO:52), specific for ME180;xe2x80x83xe2x80x83(x)
and the oligonucleotides complementary to these sequences.
In a preferred embodiment of the method, said HPV type-specific oligonucleotide probes are applied in the form of two separate probe mixtures, one mixture containing probes specific for HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 54, 56 and 58, and not containing probes specific for HPV types 6, 11, 34, 40, 42, 43 and 44, and the other mixture containing probes specific for HPV types 6, 11, 34, 40, 42, 43 and 44, and not containing probes specific for HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 54, 56 and 58.
According to the above preferred embodiment, the HPV high risk probe mixture contains up to 12 different HPV-specific oligonucleotide probes, preferably all 12. The mixture is not necessarily complete, however, and it may be advisable to add type-specific probes for other high risk HPV types, such as HPV 59, HPV66 and ME180. The preferred HPV low risk probe mixture as shown above contains up to 7 different HPV-specific oligonucleotide probes, preferably all 7. The composition of this low risk HPV probe mixture is certainly incomplete, however, because only those HPV types are included which are frequently present in the Dutch population. Therefore, both the high risk and the low risk probe cocktails need to be supplemented in future when new identified high risk HPVs and frequently present low risk HPVs are found. Especially the cocktail probe detecting the high risk HPVs is very important for cervical cancer screening and should therefore be as complete as possible.
It is preferred but not required to use probe mixtures. Instead, it is also possible to use the individual probes separately. Although it seems most practical to compose one high risk and one low risk cocktail probe, it is also possible to prepare e.g. two different high risk probe mixtures which together cover all high risk HPV types. The same can be done with the low risk cocktail probe: it can be divided over two (or more) different probe mixtures.
Although the probes can carry any suitable probe label, such as radioactive labels, enzyme labels, fluorescent labels, etc., they preferably comprise digoxygenine as a label.
The invention is furthermore embodied in a HPV type-specific oligonucleotide probe useful in a method as described above, the oligonucleotide of the probe being selected from the group consisting of:
5xe2x80x2-ATCCGTAACTACATCTTCCACATACACCAA-3xe2x80x2, (SEQ ID NO:29), specific for HPV-6;xe2x80x83xe2x80x83(a)
5xe2x80x2-ATCTGTGTCTAAATCTGCTACATACACTAA-3xe2x80x2, (SEQ ID NO:30), specific for HPV-11;xe2x80x83xe2x80x83(b)
5xe2x80x2-GTCATTATGTGCTGCCATATCTACTTCAGA-3xe2x80x2, (SEQ ID NO:31), specific for HPV-16;xe2x80x83xe2x80x83(c)
5xe2x80x2-TGCTTCTACACAGTCTCCTGTACCTGGGCA-3xe2x80x2, (SEQ ID NO:32), specific for HPV-18;xe2x80x83xe2x80x83(d)
5xe2x80x2-AGTACATTATCTGCAGCATCTGCATCCACT-3xe2x80x2, (SEQ ID NO:33), specific for HPV-26;xe2x80x83xe2x80x83(e)
5xe2x80x2-TGTTTGTGCTGCAATTGCAAACAGTGATAC-3xe2x80x2, (SEQ ID NO:34), specific for HPV-31;xe2x80x83xe2x80x83(f)
5xe2x80x2-TTTATGCACACAAGTAACTAGTGACAGTAC-3xe2x80x2, (SEQ ID NO:35), specific for HPV-33;xe2x80x83xe2x80x83(g)
5xe2x80x2-TACACAATCCACAAGTACAAATGCACCATA-3xe2x80x2, (SEQ ID NO:36), specific for HPV-34;xe2x80x83xe2x80x83(h)
5xe2x80x2-GTCTGTGTGTTCTGCTGTGTCTTCTAGTGA-3xe2x80x2, (SEQ ID NO:37), specific for HPV-35;xe2x80x83xe2x80x83(i)
5xe2x80x2-TCTACCTCTATAGAGTCTTCCATACCTTCT-3xe2x80x2, (SEQ ID NO:38), specific for HPV-39;xe2x80x83xe2x80x83(j)
5xe2x80x2-GCTGCCACACAGTCCCCCACACCAACCCCA-3xe2x80x2, (SEQ ID NO:39), specific for HPV-40;xe2x80x83xe2x80x83(k)
5xe2x80x2-CTGCAACATCTGGTGATACATATACAGCTG-3xe2x80x2, (SEQ ID NO:40), specific for HPV-42;xe2x80x83xe2x80x83(l)
5xe2x80x2-TCTACTGACCCTACTGTGCCCAGTACATAT-3xe2x80x2, (SEQ ID NO:41), specific for HPV-43;xe2x80x83xe2x80x83(m)
xe2x80x835xe2x80x2-GCCACTACACAGTCCCCTCCGTCTACATAT-3xe2x80x2, (SEQ ID NO:42), for HPV-44;xe2x80x83xe2x80x83(n)
5xe2x80x2-ACACAAAATCCTGTGCCAAGTACATATGAC-3xe2x80x2, (SEQ ID NO:43), for HPV-45;xe2x80x83xe2x80x83(o)
5xe2x80x2-AGCACTGCCACTGCTGCGGTTTCCCCAACA-3xe2x80x2, (SEQ ID NO:44), for HPV-51;xe2x80x83xe2x80x83(p)
5xe2x80x2-TGCTGAGGTTAAAAAGGAAAGCACATATAA-3xe2x80x2, (SEQ ID NO:45), for HPV-52;xe2x80x83xe2x80x83(q)
5xe2x80x2-TACAGCATCCACGCAGGATAGCTTTAATAA-3xe2x80x2, (SEQ ID NO:46), for HPV-54;xe2x80x83xe2x80x83(r)
5xe2x80x2-GTACTGCTACAGAACAGTTAAGTAAATATG-3xe2x80x2, (SEQ ID NO:47), for HPV-56;xe2x80x83xe2x80x83(s)
5xe2x80x2-ATTATGCACTGAAGTAACTAAGGAAGGTAC-3xe2x80x2, (SEQ ID NO:48), for HPV-58;xe2x80x83xe2x80x83(t)
5xe2x80x2-TCTACTACTGCTTCTATTCCTAATGTATAC-3xe2x80x2, (SEQ ID NO:49), for HPV-59;xe2x80x83xe2x80x83(u)
5xe2x80x2-TACTGCTACATCCCCCCCTGTATCTGAATA-3xe2x80x2, (SEQ ID NO:50), for HPV-61;xe2x80x83xe2x80x83(v)
5xe2x80x2-TATTAATGCAGCTAAAAGCACATTAACTAA-3xe2x80x2, (SEQ ID NO:51), for HPV-66;xe2x80x83xe2x80x83(w)
5xe2x80x2-TCTACTACTACTGAATCAGCTGTACCAAAT-3xe2x80x2, (SEQ ID NO:52), for ME180;xe2x80x83xe2x80x83(x)
and the oligonucleotides complementary to these sequences.
The invention also includes a HPV high risk cocktail probe which is a mixture of oligonucleotide probes useful in the above method, said mixture containing probes specific for HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 54, 56 and 58, and not containing probes specific for HPV types 6, 11, 34, 40, 42, 43 and 44. As discussed above, the high risk cocktail probe is preferably as complete as possible and therefore preferably contains probes for further high risk HPV types. The high risk probe mixture may be presented as one complete mixture, or alternatively as two or more different probe mixtures which together cover the high risk HPV types as completely as possible.
The invention also includes a HPV low risk cocktail probe which is a mixture of oligonucleotide probes useful in the above method, said mixture containing probes specific for HPV types 6, 11, 34, 40, 42, 43 and 44, and not containing probes specific for HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 54, 56 and 58. The observations made above in connection with the high risk cocktail probe are valid also for the low risk cocktail probe.
The invention also includes an assembly of a HPV high risk cocktail probe and a HPV low risk cocktail probe, said assembly comprising a mixture containing probes specific for HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 54, 56 and 58, and not containing probes specific for HPV types 6, 11, 34, 40, 42, 43 and 44, said assembly further comprising a mixture containing probes specific for HPV types 6, 11, 34, 40, 42, 43 and 44, and not containing probes specific for HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 54, 56 and 58.